Evolution Of Flowering Plants Research Articles

Effect of the suppression of BpAP1 on the expression of lignin related genes in birch

Lignin is an integral part of secondary cell walls in plants and plays important roles in maintaining the strength of stems, enhancing transport ability of stems, and providing resistance to multiple stresses. Lignin biosynthesis has become one of the hotspots in molecular forest biology research. The AP1 transcription factor plays important roles in plant flower development. However, in this study, suppression of BpAP1 altered the transcription profiles of white birch and RNA-seq was used to find that suppression of BpAP1 changed the expression of lignin pathway-related genes; C4H/CYP73A, POD were down-regulated and HCT, CCoAOMT, REF1 and CAD were up-regulated. Cell walls of the suppressed transgenic birch were significantly thinner than the wild type of birch, and BpAP1- repressed birch contained less lignin. In addition to regulation of floral development, BpAP1 might play a role in regulating the expression of genes in lignin biosynthesis of birch. This study could provide a new insight into the function of AP1 genes in woody species.

Gibberellin Metabolism in Flowering Plants: An Update and Perspectives.

In plants, gibberellins (GAs) play important roles in regulating growth and development. Early studies revealed the large chemodiversity of gibberellins in plants, but only GA1, GA3, GA4, and GA7 show biological activity that controls plant development. However, the elucidation of the GA metabolic network at the molecular level has lagged far behind the chemical discovery of GAs. Recent advances in downstream GA biosynthesis (after GA12 formation) suggest that species-specific gibberellin modifications were acquired during flowering plant evolution. Here, we summarize the current knowledge of GA metabolism in flowering plants and the physiological functions of GA deactivation, with a focus on GA 13 hydroxylation. The potential applications of GA synthetic biology for plant development are also discussed.

Water lily (Nymphaea thermarum) genome reveals variable genomic signatures of ancient vascular cambium losses

For more than 225 million y, all seed plants were woody trees, shrubs, or vines. Shortly after the origin of angiosperms ∼140 million y ago (MYA), the Nymphaeales (water lilies) became one of the first lineages to deviate from their ancestral, woody habit by losing the vascular cambium, the meristematic population of cells that produces secondary xylem (wood) and phloem. Many of the genes and gene families that regulate differentiation of secondary tissues also regulate the differentiation of primary xylem and phloem, which are produced by apical meristems and retained in nearly all seed plants. Here, we sequenced and assembled a draft genome of the water lily Nymphaea thermarum, an emerging system for the study of early flowering plant evolution, and compared it to genomes from other cambium-bearing and cambium-less lineages (e.g., monocots and Nelumbo). This revealed lineage-specific patterns of gene loss and divergence. Nymphaea is characterized by a significant contraction of the HD-ZIP III transcription factors, specifically loss of REVOLUTA, which influences cambial activity in other angiosperms. We also found the Nymphaea and monocot copies of cambium-associated CLE signaling peptides display unique substitutions at otherwise highly conserved amino acids. Nelumbo displays no obvious divergence in cambium-associated genes. The divergent genomic signatures of convergent loss of vascular cambium reveals that even pleiotropic genes can exhibit unique divergence patterns in association with independent events of trait loss. Our results shed light on the evolution of herbaceousness-one of the key biological innovations associated with the earliest phases of angiosperm evolution.

Comparative metabolomics and transcriptomics of pistils, stamens and pistilloid stamens widen key knowledge of pistil and stamen development in wheat.

To examine the role of metabolites in wheat stamen and pistil development, metabolomic analyses of pistilloid stamens (PS), pistils (P), and stamens (S) from a novel wheat mutant homologous transformation sterility-1 (HTS-1) and controls from their sib-line CSTP were conducted using base gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). Then, the metabolomic data were integrated with previously published transcriptomic data and analysed. In total, 141 annotated metabolites were determined from P, PS and S tissues by comparison with reference standards. A total of 90, 93 and 18 different metabolites were identified in S vs. PS, S vs. P and P vs. PS, respectively. Among the different metabolites, 80 may be associated with stamen and pistil growth. Using integration evaluations of both the previous transcriptome data and the 80 various metabolites, we found two perturbed pathways that significantly affect flower development in plants, namely, the phenylpropanoid biosynthesis and cysteine and methionine metabolism. The ethylene synthesis pathway, one key branch of the cysteine and methionine metabolic pathways, could have a pivotal role in pistillody growth involving HTS-1. We found two key enzyme genes in the ethylene synthesis pathway (the SAM synthase gene and the ACC synthase gene) that have higher expression levels in stamens than in pistilloid stamens or pistils. We speculate, that the decrease in ethylene content during stamen development leads to pistillody traits in HTS-1. This study helps elucidate the molecular mechanisms underlying stamen and pistil growth in wheat.

Discovering and Constructing ceRNA-miRNA-Target Gene Regulatory Networks during Anther Development in Maize.

The “competing endogenous RNA (ceRNA) hypothesis” has recently been proposed for a new type of gene regulatory model in many organisms. Anther development is a crucial biological process in plant reproduction, and its gene regulatory network (GRN) has been gradually revealed during the past two decades. However, it is still unknown whether ceRNAs contribute to anther development and sexual reproduction in plants. We performed RNA and small RNA sequencing of anther tissues sampled at three developmental stages in two maize lines. A total of 28,233 stably transcribed loci, 61 known and 51 potentially novel microRNAs (miRNAs) were identified from the transcriptomes. Predicted ceRNAs and target genes were found to conserve in sequences of recognition sites where their corresponding miRNAs bound. We then reconstructed 79 ceRNA-miRNA-target gene regulatory networks consisting of 51 known miRNAs, 28 potentially novel miRNAs, 619 ceRNA-miRNA pairs, and 869 miRNA-target gene pairs. More than half of the regulation pairs showed significant negative correlations at transcriptional levels. Several well-studied miRNA-target gene pairs associated with plant flower development were located in some networks, including miR156-SPL, miR159-MYB, miR160-ARF, miR164-NAC, miR172-AP2, and miR319-TCP pairs. Six target genes in the networks were found to be orthologs of functionally confirmed genes participating in anther development in plants. Our results provide an insight that the ceRNA-miRNA-target gene regulatory networks likely contribute to anther development in maize. Further functional studies on a number of ceRNAs, miRNAs, and target genes will facilitate our deep understanding on mechanisms of anther development and sexual plants reproduction.

Origin of angiosperms and the puzzle of the Jurassic gap

Angiosperms are by far the most species-rich clade of land plants, but their origin and early evolutionary history remain poorly understood. We reconstructed angiosperm phylogeny based on 80 genes from 2,881 plastid genomes representing 85% of extant families and all orders. With a well-resolved plastid tree and 62 fossil calibrations, we dated the origin of the crown angiosperms to the Upper Triassic, with major angiosperm radiations occurring in the Jurassic and Lower Cretaceous. This estimated crown age is substantially earlier than that of unequivocal angiosperm fossils, and the difference is here termed the 'Jurassic angiosperm gap'. Our time-calibrated plastid phylogenomic tree provides a highly relevant framework for future comparative studies of flowering plant evolution.

RAD sequencing rejects a long‐distance disjunction in Stellaria (Caryophyllaceae) and yields support for a new southern Rocky Mountains endemic

Whereas the eastern North American–eastern Asian floristic connection represents one of the most widely studied biogeographical relationships in flowering plant evolution, connections between western North America and Asia have been comparatively rarely investigated, especially through genetic approaches. Stellaria irrigua is one of several plants that has been treated as an exceptionally dramatic example of a disjunction between floristically similar, high alpine biotas of the southern Rocky Mountains and south‐central Siberia. We here employ numerous new field collections and ddRADseq data to test the hypothesis that S. irrigua—a species that has been known for over 180 years—represents a long‐distance disjunction between the southern Rocky Mountains and central Asia. Extensive fieldwork, review and perusal of herbarium materials, and phylogenomic analyses indicate that S. irrigua is broadly distributed across an amphi‐Beringian arc extending from southern and central Asia, east through Beringia, and south throughout mountainous regions of western North America. Sampled Asian populations formed two clades, and North American individuals all formed a clade embedded within this broader Asian lineage. Stellaria irrigua is, however, rendered non‐monophyletic by a lineage that is embedded within the North American populations and is ecologically and morphologically distinctive from S. irrigua. The identity of this newly recognized lineage, which was in prior works attributed to S. irrigua, has been confused since plants of the former were first collected in the San Juan Mountains of Colorado in the late 1800s under Arenaria and Alsine. We provide a new name for this taxon, Stellaria sanjuanensis, a charismatic starwort of dry alpine scree slopes of the southern Rocky Mountains. Additionally, two lectotypes are designated, one holotype and one isotype are identified, and two new synonymies are proposed, to help stabilize the taxonomy and nomenclature of this long‐confused species complex. A key to the starworts of the southern Rocky Mountains is also provided, and Stellaria alsine is reported as new to the region.

Transgenerational effects of inter-ploidy cross direction on reproduction and F2 seed development of Arabidopsis thaliana F1 hybrid triploids

Key messageReproduction in triploid plants is important for understanding polyploid population dynamics. We show that genetically identical reciprocal F1 hybrid triploids can display transgenerational epigenetic effects on viable F2 seed development.The success or failure of reproductive outcomes from intra-species crosses between plants of different ploidy levels is an important factor in flowering plant evolution and crop breeding. However, the effects of inter-ploidy cross directions on F1 hybrid offspring fitness are poorly understood. In Arabidopsis thaliana, hybridization between diploid and tetraploid plants can produce viable F1 triploid plants. When selfed, such F1 triploid plants act as aneuploid gamete production “machines” where the vast majority of gametes generated are aneuploid which, following sexual reproduction, can generate aneuploid swarms of F2 progeny (Henry et al. 2009). There is potential for some aneuploids to cause gametophyte abortion and/or F2 seed abortion (Henry et al. 2009). In this study, we analyse the reproductive success of 178 self-fertilized inter-accession F1 hybrid triploids and demonstrate that the proportions of aborted or normally developed F2 seeds from the selfed F1 triploids depend upon a combination of natural variation and cross direction, with strong interaction between these factors. Single-seed ploidy analysis indicates that the embryonic DNA content of phenotypically normal F2 seeds is highly variable and that these DNA content distributions are also affected by genotype and cross direction. Notably, genetically identical reciprocal F1 hybrid triploids display grandparent-of-origin effects on F2 seed set, and hence on the ability to tolerate aneuploidy in F2 seed. There are differences between reciprocal F1 hybrid triploids regarding the proportions of normal and aborted F2 seeds generated, and also for the DNA content averages and distributions of the F2 seeds. To identify genetic variation for tolerance of aneuploidy in F2 seeds, we carried out a GWAS which identified two SNPs, termed MOT and POT, which represent candidate loci for genetic control of the proportion of normal F2 seeds obtained from selfed F1 triploids. Parental and grandparental effects on F2 seeds obtained from selfed F1 triploids can have transgenerational consequences for asymmetric gene flow, emergence of novel genotypes in polyploid populations, and for control of F2 seed set in triploid crops.

Identification of Two GLOBOSA-like MADS-box Genes in Tea Plant (Camellia sinensis [L.] O. Kuntze)

Tea plant (Camellia sinensis [L.] O. Kuntze) is a woody crop of high economic importance worldwide; however, information on the molecular mechanisms underlying the regulation of flower development in this species is limited. In the present study, two GLOBOSA (GLO) -like MADS-box genes, CsGLO1 and CsGLO2, were isolated from C. sinensis 'Ziyangzhong' and were characterized to elucidate their roles in flower development. We found that CsGLOl and CsGLO2 are nuclear-localized transcription factors without transactivation ability but with a robust interaction. They have similar patterns of expression, both mainly restricted to petals and stamens. Moreover, ectopic expression of either CsGLO1 or CsGLO2 in Arabidopsis thaliana resulted in a partial conversion of sepals to petals, suggesting full GLOBOSA functional activity. Our results indicate that CsGLO1 and CsGLO2 paralogs might redundantly contribute to petal and stamen, providing the first insight into their role in tea plant flower development.

Apomixis in flowering plants: Developmental and evolutionary considerations.

Apomixis refers to a set of reproductive mechanisms that invariably rely on avoiding meiotic reduction and fertilization of the egg cell to generate clonal seeds. After having long been considered a strictly asexual oddity leading to extinction, the integration of more than 100 years of embryological, genetic, molecular, and ecological research has revealed apomixis as a widely spread component of the dynamic processes that shape flowering plant evolution. Apomixis involves several flexible and versatile developmental pathways that can be combined within the ovule to produce offspring. Here we review the large body of classic and contemporaneous contributions that have addressed unreduced gamete formation, haploid induction, and parthenogenesis in flowering plants. We emphasize similarities and differences between sexual and apomictic reproduction, and highlight their implications for the evolutionary emergence of asexual reproduction through seeds. On the basis of these comparisons, we propose a model that associates the developmental origin of apomixis to a dynamic epigenetic landscape, in which environmental fluctuations reversibly influence female reproductive development through mechanisms of hybridization and polyploidization.

Modulation of Auxin Levels in Pollen Grains Affects Stamen Development and Anther Dehiscence in Arabidopsis.

Auxin regulates diverse aspects of flower development in plants, such as differentiation of the apical meristem, elongation of the stamen, and maturation of anthers and pollen. It is known that auxin accumulates in pollen, but little information regarding the biological relevance of auxin in this tissue at different times of development is available. In this work, we manipulated the amount of free auxin specifically in developing pollen, using transgenic Arabidopsis lines that express the bacterial indole-3-acetic acid-lysine synthetase (iaaL) gene driven by a collection of pollen-specific promoters. The iaaL gene codes for an indole-3-acetic acid-lysine synthetase that catalyzes the conversion of free auxin into inactive indole-3-acetyl-l-lysine. The transgenic lines showed several abnormalities, including the absence of short stamina, a diminished seed set, aberrant pollen tubes, and perturbations in the synchronization of anther dehiscence and stamina development. This article describes the importance of auxin accumulation in pollen and its role in stamina and anther development.

An in silico comparative genomic report on transcription factors in three Arabidopsis species

Transcription factors (TF) are the elements, which regulate gene expression. Regulatory function of TFs play an important role in plant biological processes and mechanisms. They may interconnect with other transcription factors or functional genes to modulate their expression in response to an internal/external factor like life cycle stage, growth, development and stress. Arabidopsis is the well-known and the most used model organism. Transcription factors of three Arabidopsis species including A. halleri, A. lyrata and A. thaliana, were compared. basic/helix-loop-helix (bHLH) with 220 TFs was the most abundant family among three Arabidopsis species while MYB and MYB related families considering as a whole group were more than bHLH with 308 TFs. No STERILE APETALA (SAP) TF homolog was found for A.halleri. The common transcription factors among three species were 4,172 grouped in 1,212 clusters. The species-specific clustered TFs were 12, 30 and 58 for A. halleri, A. lyrata and A. thaliana respectively. Eight hundred ninety two single-copy gene clusters those have one gene copy from each species, i.e. 2,676 genes, were listed. Four hundred forty five TF singletons were not clustered and are unique among three species. For clustered TF belonging to each species, GO terms and SwissProt hits showed that A. halleri has two species-specific TFs involved in heavy metal response including Zinc finger protein AZF2 and two-component response regulator ARR11 while for A. lyrata specific TFs are involved in stress response and plant development. A. thaliana specific clustered TFs work on plant flower development and acclimation.

An in silico comparative genomic report on transcription factors in three Arabidopsis species

Transcription factors (TF) are the elements, which regulate gene expression. Regulatory function of TFs play an important role in plant biological processes and mechanisms. They may interconnect with other transcription factors or functional genes to modulate their expression in response to an internal/external factor like life cycle stage, growth, development and stress. Arabidopsis is the well-known and the most used model organism. Transcription factors of three Arabidopsis species including A. halleri, A. lyrata and A. thaliana, were compared. basic/helix-loop-helix (bHLH) with 220 TFs was the most abundant family among three Arabidopsis species while MYB and MYB related families considering as a whole group were more than bHLH with 308 TFs. No STERILE APETALA (SAP) TF homolog was found for A.halleri. The common transcription factors among three species were 4,172 grouped in 1,212 clusters. The species-specific clustered TFs were 12, 30 and 58 for A. halleri, A. lyrata and A. thaliana respectively. Eight hundred ninety two single-copy gene clusters those have one gene copy from each species, i.e. 2,676 genes, were listed. Four hundred forty five TF singletons were not clustered and are unique among three species. For clustered TF belonging to each species, GO terms and SwissProt hits showed that A. halleri has two species-specific TFs involved in heavy metal response including Zinc finger protein AZF2 and two-component response regulator ARR11 while for A. lyrata specific TFs are involved in stress response and plant development. A. thaliana specific clustered TFs work on plant flower development and acclimation.

Macroevolutionary Patterns of Flowering Plant Speciation and Extinction.

Species diversity is remarkably unevenly distributed among flowering plant lineages. Despite a growing toolbox of research methods, the reasons underlying this patchy pattern have continued to perplex plant biologists for the past two decades. In this review, we examine the present understanding of transitions in flowering plant evolution that have been proposed to influence speciation and extinction. In particular, ploidy changes, transitions between tropical and nontropical biomes, and shifts in floral form have received attention and have offered some surprises in terms of which factors influence speciation and extinction rates. Mating systems and dispersal characteristics once predominated as determining factors, yet recent evidence suggests that these changes are not as influential as previously thought or are important only when paired with range shifts. Although range extent is an important correlate of speciation, it also influences extinction and brings an applied focus to diversification research. Recent studies that find that past diversification can predict present-day extinction risk open an exciting avenue for future research to help guide conservation prioritization.

Identification and expression analysis of 11 MADS-box genes in peach (Prunus persica var. nectarina ‘Luxing’)

ABSTRACTMADS-box genes play a central role in the development of flowers in plants. In this study, 11 PpMADSs were isolated from ‘Luxing’ peach, and the expression levels were detected in different tissues and fruit development. Eleven PpMADSs, designated as PpMADS13, 14, 18, 23, 24, 25, 32, 33, 34, 35, and 39 were isolated. Phylogenetic analysis revealed that PpMADS13, 14, and 18 belonged to SVP, AGL15, and MIKC* group respectively; PpMADS23, 24, 25, and 39 were in the Mα group; PpMADS32, 33, 34, and 35 belonged to the Mγ group. Predictions from subcellular localization showed that 10 PpMADS were located in the nucleus. RT-PCR revealed that PpMADS13 was expressed in stems, leaves, and during fruit development (70d); PpMADS14 was expressed in sepals, stamens, petals, and during flower development; PpMADS18 was expressed in roots, stems, leaves, sepals, ovaries, stamens, petals, and during flower and fruit development; all MADS-box genes (expect for PpMADS33) in the Mα and Mγ group were expressed in roots, stems, leaves, sepals, ovaries, stamens, petals, and during flower development; few genes were expressed during fruit development. These results indicated that PpMADS may play a crucial regulatory role in vegetative growth and development processes of flowers and fruits in peaches.

Pollen of colombian magnolias

La familia Magnoliaceae ha sido de gran interés para los científicos que buscan entender la evolución de las plantas en flor. Los caracteres morfológicos han sido investigados en varios estudios, especialmente en China, el país con mayor número de especies. Colombia, con 36 especies, tiene el mayor número de especies en América del Sur. A pesar de la importancia evolutiva de la familia y las amenazas significativas a las especies supervivientes, todavía falta información sobre las Magnoliaceae colombianas debido a la falta de investigaciones. En este artículo se describe la morfología del polen de catorce especies de Magnolia de Colombia con base en su tamaño, forma, aberturas, exina y escultura. Los granos de polen de material fresco y colecciones de herbario fueron estudiados usando métodos de acetólisis estándar, y las descripciones morfológicas fueron elaboradas a partir de las observaciones de microscopía de luz y microscopía electrónica de barrido. En este estudio los granos de polen se consideraron grandes (de 68,2 a 115 μm en el eje más largo y de 41,4 a 69,3 μm en el eje más corto), en forma de barco y anasulcado. En las especies analizadas se encontraron patrones de escultura en exina tales como rugoso, psilado, perforado y combinaciones de estos tipos. Aunque existe una gran uniformidad en la forma de los granos de polen, se encontraron algunas diferencias entre las especies, no sólo en tamaño sino también en ornamentación. La alta uniformidad de la morfología del polen entre las especies colombianas apoya la clasificación más reciente de las magnolias americanas.

Genomic signature of successful colonization of Eurasia by the allopolyploid shepherd's purse (Capsella bursa-pastoris).

Polyploidization is a dominant feature of flowering plant evolution. However, detailed genomic analyses of the interpopulation diversification of polyploids following genome duplication are still in their infancy, mainly because of methodological limits, both in terms of sequencing and computational analyses. The shepherd's purse (Capsella bursa-pastoris) is one of the most common weed species in the world. It is highly self-fertilizing, and recent genomic data indicate that it is an allopolyploid, resulting from hybridization between the ancestors of the diploid species Capsella grandiflora and Capsella orientalis. Here, we investigated the genomic diversity of C.bursa-pastoris, its population structure and demographic history, following allopolyploidization in Eurasia. To that end, we genotyped 261 C.bursa-pastoris accessions spread across Europe, the Middle East and Asia, using genotyping-by-sequencing, leading to a total of 4274 SNPs after quality control. Bayesian clustering analyses revealed three distinct genetic clusters in Eurasia: one cluster grouping samples from Western Europe and Southeastern Siberia, the second one centred on Eastern Asia and the third one in the Middle East. Approximate Bayesian computation (ABC) supported the hypothesis that C.bursa-pastoris underwent a typical colonization history involving low gene flow among colonizing populations, likely starting from the Middle East towards Europe and followed by successive human-mediated expansions into Eastern Asia. Altogether, these findings bring new insights into the recent multistage colonization history of the allotetraploid C.bursa-pastoris and highlight ABC and genotyping-by-sequencing data as promising but still challenging tools to infer demographic histories of selfing allopolyploids.

Late Aptian angiosperm pollen grains from Patagonia: Earliest steps in flowering plant evolution at middle latitudes in southern South America

The angiosperm pollen record from the Anfiteatro de Ticó and Punta del Barco formations (Baqueró Group) is reported. The relevance of these floras is that they are accurately dated as late Aptian, and one of the oldest floras southern South America with fossil angiosperms. Twelve samples were studied, showing Clavatipollenites and Retimonocolpites as dominant types. A new species, Jushingipollis ticoensis sp. nov., is proposed. A doubtful angiosperm pollen grain, Lethomasites sp., is also identified and described. A multivariate analysis of similarities between different Early Cretaceous angiosperm pollen assemblages suggests that the Baqueró Group assemblages have great similarities with other coeval units from Argentina, Australia and United States, which were located in a similar paleolatitude.

Heat mediated silencing of MdTFL1 genes in apple (Malus × domestica)

The apple TFL1 genes, MdTFL1-1 and MdTFL1-2, were tried to be silenced using the opposing dual-promoter gene construct 620p9U10-HSP::MdTFL1-HSP containing a 323 bp fragment of MdTFL1-1 flanked by two inverted sequences of the Gmhsp17.5-E heat-inducible promoter. This construct was used for transformation experiments with the apple cultivars ‘Gala’ and ‘Pinova’. Three (T967, T973, and T985) and two (T976 and T987) transgenic lines were obtained for ‘Pinova’ and ‘Gala’, respectively. Single heat shock treatments at 42 °C for 1 and 2 h, respectively, were applied to plants of transgenic lines and non-transformed controls grown in vitro or in the glasshouse. Heat shock treatments induced a short-term activation of the transgene construct. Silencing of MdTFL1-1 and MdTFL1-2 was detected in transgenic plants, but also in non-transformed controls, highlighting a similar heat response. The transcription of both MdTFL1 genes was repressed for 24 h post heat shock induction. In parallel, we treated shoots of glasshouse-grown plants at 42 °C for 2 h daily for the duration of 28 days. This treatment resulted in silencing of MdTFL1 in leaves of transgenic and non-transformed plants. However, repeated heat shock treatments failed to induce flower development in plants. Notwithstanding, we recorded a number of deformed leaves on transgenic shoots in comparison to non-transformed ones. This has lead to the conclusion that the transgene construct is functional in down-regulation of TFL1 homologs. The heat shock treatments demonstrated a major role of both MdTFL1 genes in plant’s heat response.

Genome-wide identification, expression profiling, and SSR marker development of the bZIP transcription factor family in Medicago truncatula

The basic leucine zipper (bZIP) transcription factors have been shown to play great roles in pathogen defense, light and stress signalling, seed maturation and flower development in plants. However, no reports about this transcription factor family have been reported in grass species, including the model dicotyledonous grass species Medicago truncatula. In the present study, we have identified 75 bZIP transcription factor-encoding genes in the M. truncatula genome. The phylogenetic analysis of these bZIP protein sequences permits classification of them into 12 groups. The chromosome distribution analyses suggesting that bZIP genes in M. truncatula are not recently generated by gene duplication. The results of the identification of intron/exon structural and conserved motifs showed that there is a high group-specificity in all MtbZIP genes. Expression of bZIP transcription factor-encoding genes indicates specific or coexpression patterns of MtbZIP genes involved in organ development, drought-responses and salt-responses. In addition, 104 SSRs specific to 53 MtbZIP genes were detected and 97 primer pairs were developed. The validation of 30 randomly selected primer pairs in 20 alfalfa cultivars showed 17 of them were true-to-type SSR loci and highly transferable among alfalfa cultivars. These results will be important for further cloning and functional characterization of bZIP transcription factors in M. truncatula.